The IL-2 receptor and related cytokine/cytokine receptor systems are being studied to understand the T cell immune response in normal and neoplastic cells. After T-cell activation, the magnitude and duration of the response is controlled by IL-2, levels of IL-2 receptors, and the time course of their induction. IL-2Ra expression is very high in cells infected with HTLV-I, the cause of adult T cell leukemia (ATL). There are 3 chains of the receptor: IL-2Ra, IL-2Rb, and gc, with IL-2Ra and IL-2Rb being regulated at the level of transcription. gc is shared by the IL-4, IL-7, IL-9, IL-15, and IL-21 receptors and is mutated in XSCID. We study the signals induced by these cytokines, particularly STAT proteins and the mechanisms by which they regulate target genes. Given our prior data that Stat5a or Stat5b transgenic mice develop tumors, consistent with STAT5 being implicated in malignant transformation and elevated in a range of human tumors, this has relevance for both normal and pathological states. Moreover, humans and mice with defective STAT protein expression have immunological defects. T helper cell differentiation is critical for normal immune responses, with Th1 differentiation important for host defense to viruses and other intracelllular pathogens, Th2 differentiation vital in allergic disorders/helminths, and Th17 differentiation vital in inflammatory disorders, including psoriasis and inflammatory bowel disease. We previously showed that IL-2 is important for Th2 differentiation and that IL-2 induces IL-4 receptor expression in a STAT5-dependent manner and controls priming of cells for Th2 differentiation. Moreover, using genome-wide chromatin immunoprecipitation coupled to DNA sequencing (ChIP-Seq) analysis, we previously found broad regulation of Th2 differentiation via STAT5A and STAT5B. We previously extended these findings by showing that IL-2 via STAT5 induces IL-12Rb2, which is critical for Th1 differentiation. We also showed that IL-2 via STAT5 also regulates T-bet. Interestingly, IL-2 also inhibits expression of IL-6Ra and gp130, helping to explain the inhibition of Th17 differentiation. We also had reported a critical role of IL-2 in Th9 differentiation, with a direct effect of IL-2 on Th9 differentiation via its induction of STAT5 binding to the Il9 promoter and that IL-2 and IL-21 had opposing actions in Th9 differentiation based on induction of BCL6 by IL-21 but repression by IL-2. We previously collaborated with Dr. K. Christopher Garcia (Stanford), comparing IL-2 and IL-15 receptor structures, providing mechanistic and structural insights into the functional differences between IL-2 and IL-15. We had extended this collaboration and generated novel IL-2 variants, which represent the first partial agonists for a type 1 cytokine. These next-generation IL-2 variants function as receptor signaling clamps, retaining high affinity for IL-2Rb, inhibiting binding of endogenous IL-2, but their interaction with gc was weakened, attenuating IL-2Rb/gc heterodimerization. We previously showed that one variant, denoted H9-RETR, could prolong survival in a model of graft-versus-host disease and blocked spontaneous proliferation of smoldering adult T cell leukemia (ATL) T cells. This receptor-clamping approach might be a general mechanism-based strategy with applications to other type 1 cytokines as well. During the past year, we have continued our study of these molecules, studying new variants and extending collaborative pre-clinical studies of these molecules. IL-21 has broad actions on T- and B-cells, but its actions in innate immunity are poorly understood. We previously reported that IL-21 induced apoptosis of conventional dendritic cells (cDCs) occurs via STAT3 and Bim, and this was inhibited by granulocyte-macrophage colony-stimulating factor (GM-CSF). ChIP-Seq analysis had revealed genome-wide binding competition between GM-CSF-induced STAT5 and IL-21-induced STAT3, and moreover, we previously elucidated roles for STAT1 vs. STAT3 in IL-21 signaling in T cells. Previously, we also demonstrated that IL-21 regulates expression of the Prdm1 gene that encodes BLIMP1 via a response element that depends on STAT3 and IRF4 and subsequently discovered that in contrast to its known ability to cooperate with PU.1 in B cells to act via Ets-IRF composite elements (EICEs), IRF4 cooperates with BATF/JUN family proteins to act via novel AP1-IRF composite elements (AICEs) in T cells, as well as in B cells. We demonstrated cooperative regulation of important genes via these AICEs and cooperative binding of IRF4, BATF, and JUN family proteins, with markedly diminished IRF4 binding in Batf-deficient cells and markedly diminished BATF binding in Irf4-deficient cells. We have continued studies of AICEs and IRF4/BATF/JUN/STAT3 complexes, reporting a collaborative study with Nick Restifo (NCI) that BACH2 regulates CD8 T cell differentiation by controlling access of AP1 factors to enhancer. We also studied the expression of IRF8 as a protective factor for H. Pylori infection with H.C. Morse (NIAID) and contributed to a study by Axel Kallies (WEHI) showing that IL-2 and IL-12 together with BLIMP1 and TBET control effector CD8 T cell differentiation. Moreover, we also contributed to a study by the on the relationship of mitochondria member potential as an identifier of high stemness for cellular therapy. Previously, we studied the biological significance of STAT5 tetramerization in vivo by generating mice expressing mutant forms of STAT5A and STAT5B that could form dimers but not tetramers. This past year we reported intricate modeling of the 3-dimensional structure of the tetramer, providing new insights into its modeling. We also substantially extended our analysis of the basis for defective NK cell development in the double knockin mice. Moreover, we generated mice with other mutations in the Stat5a and Stat5b genes and also have furthered studied STAT biology using next generation sequencing approaches. In other studies, having reported the biological roles of Egr1 and Egr2 in T cells and their relationship to IL-2 signaling, this past year we have elucidated some non-immunological roles for Egr family proteins. Overall, these studies enhance our understanding of mechanisms by which gc family cytokines regulate gene expression and biological processes and are relevant to normal and pathological immune cell function, including in allergy, autoimmunity, and cancer.